Antibodies are drawing attention as pharmaceuticals since they are highly stable in blood and have few side effects (Non-patent Documents 1 and 2). Almost all antibody pharmaceuticals currently on the market are antibodies of the human IgG1 subclass. So far, many studies have been carried out on antibody-dependent cellular cytotoxicity (hereinafter, referred to as ADCC) and complement-dependent cytotoxicity (hereinafter, referred to as CDC) which are effector functions of the IgG class antibodies; and in human IgG class, antibodies of the IgG1 subclass have been reported to have the highest ADCC activity and CDC activity (Non-patent Document 3). Furthermore, antibody-dependent cell-mediated phagocytosis (ADCP), which is phagocytosis of target cells mediated by IgG class antibodies, is also shown as one of the antibody effector functions (Non-patent Documents 4 and 5).
For an IgG antibody to exhibit ADCC, CDC, or ADCP, the antibody Fc region must bind to an antibody receptor which is present on the surface of effector cells such as killer cells, natural killer cells, and activated macrophages (hereinafter denoted as FcγR) and various complement components. In humans, the FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb isoforms have been reported as the FcγR protein family, and the respective allotypes have also been reported (Non-patent Document 6).
Enhancement of cytotoxic effector functions such as ADCC, ADCP, and CDC is drawing attention as a promising means for enhancing the anti-tumor effects of antibodies. The importance of FcγR-mediated effector functions of antibodies for their antitumor effects has been reported using mouse models (Non-patent Documents 7 and 8). Furthermore, correlation was observed between clinical effects in humans and the high-affinity polymorphic allotype (V158) and the low-affinity polymorphic allotype (F158) of FcγRIIIa (Non-patent Document 9). These reports showed that antibodies having an Fc region that has been optimized for specific FcγR-binding mediate a stronger effector function, as a result demonstrate more effective antitumor effects.
The balance between the binding activities of an antibody towards an activating receptor consisted of FcγRIa, FcγRIIa, FcγRIIIa, and FcγRIIIb, and an inhibitory receptor consisted of FcγRIIb is an important element when optimizing antibody effector function. Use of an Fc region that enhances binding activity to activating receptors and decreases binding activity to inhibitory receptors may be able to confer antibodies with optimum effector functions (Non-patent Document 10). Conversely, use of an Fc region that has sustained or decreased binding activity to activating receptors and enhanced binding activity to inhibitory receptors may be able to confer immunosuppressive effect to antibodies (Non-patent Document 11). For the binding between an Fc region and FcγR, several amino acid residues in the antibody hinge region and CH2 domain, and the sugar chain added to Asn at position 297 (EU numbering) which is bound to the CH2 domain have been shown to be important for the binding between the Fc region and FcγR (Non-patent Documents 12, 13, and 14). There has been research on Fc region variants that have various FcγR-binding properties mainly at this binding site, and Fc region variants that have higher binding activities to activating FcγR have been obtained (Patent Documents 1 and 2). For example, Lazar et al. have successfully increased human FcγRIIIa (V158) binding approximately 370-fold by substituting Ser at position 239, Ala at position 330, and Ile at position 332 (EU numbering) of human IgG1 with Asp, Leu, and Glu, respectively (Non-patent Document 15 and Patent Document 2). The ratio of FcγRIIIa-binding to FcγRIIb-binding (A/I ratio) of this variant is increased approximately 9-fold as compared to that of the wild type. Furthermore, Lazar et al. have successfully enhanced the binding to FcγRIIb approximately 430-fold (Non-patent Document 16). Shinkawa et al. have successfully enhanced the FcγRIIIa-binding approximately 100-fold by removing fucose from the sugar chain added to Asn at position 297 (EU numbering) (Non-patent Document 17).
In the above-mentioned method, the same amino acid alterations or the same sugar-chain modifications are introduced into both H-chain Fc regions of an antibody. Meanwhile, the antibody Fc region has been reported to show 1:1 binding with FcγR and recognize FcγR asymmetrically in the lower hinge and CH2 region (Non-patent Document 18). Taking into account the asymmetric interaction between the Fc region and FcγR, one may consider optimizing the interaction between IgG and FcγR more finely by introducing different alteration(s) into each of the H chains. Methods for optimizing the interaction with FcγR by asymmetrically modifying the Fc region through introduction of different alterations into each of the H-chain Fc regions of the antibody based on this idea have been reported (Patent Documents 5 and 6). However, asymmetrically-optimized Fc regions do not necessarily show excellent FcγRIIIa-binding activity in comparison to symmetrically-optimized Fc regions (Patent Document 5). Depending on the type of alterations introduced, asymmetrical optimization of the Fc region has successfully enhanced the binding to FcγRIIIa by several tens of times compared to those of a native IgG and enhanced the ADCC activity. However, on the other hand, the ADCC activity is about the same or even weaker than that of an afucosylated antibody where fucose is removed from the N-type sugar chain of both H chains, which was produced by using conventional technology (Patent Document 6).